For quantitatively determining a specific component in a biological specimens a method which comprises the steps of: reacting the biological specimen, in the presence of an electron acceptor, with an enzyme which has an ability, by the dehydrogenation reaction, to oxidize the specific component or a substance derived from the specific component, and measuring the formed reductant of the electron acceptor, by which the specific component in the biological specimen is indirectly quantitatively determined, has been known. Particularly, when a specific trace component is to be quantitatively determined, the method using a color developer having a higher color development sensitivity is also employed for detecting or quantitatively determining the formed reductant of the electron acceptor, in order to conduct the measurement with a higher sensitivity. For example, when NAD (nicotinamide adenine dinucleotide) or NADP (nicotinamide adenine dinucleotide phosphate) is used as the electron acceptor, its reductant i.e. NADH or NADPH is reacted with an electron carrier such as diaphorase, and a tetrazolium salt to form formazan for detection or quantitative determination.
However, the above quantitative determination methods are susceptible to other components in the biological specimen, for example, metal ions or metalloproteins. Particularly, when using a biological specimen in which hemoglobin, containing iron in the molecule, is included, there is a problem that accurate quantitative determination can not be conducted. For the influence of hemoglobin, there is change of absorption wavelength, which is caused by the hemoglobin being oxidized etc. Such reactions when observed during the measurement may cause errors in the measured values as a nonspecific reaction depending upon the measuring wavelength. Further, since hemoglobin protein has iron in the molecule, there is a possibility that electron transport system is inhibited.
Accordingly, various methods have been proposed as a method for avoiding the influence of hemoglobin in analysis or quantitative determination of the component contained in the test specimen. For example, JP-A-60-168050 discloses a clinical chemistry analysis method wherein a specific surfactant is added to the test specimen. JP-A-62-248500 discloses a reagent for measuring enzyme activity in serum containing thiourea. JP-A-9-84598 discloses a method wherein a reagent for measurement of enzyme activity which contains an inorganic salt sulfoxylate type reducing agent is used in measuring the activity of a specific enzyme in a specimen. JP-A-2001-292795 discloses a method wherein a nitrite is added to a reagent for measuring the concentration of an analyte contained in a hemoglobin-containing biological fluid, in order to suppress unwanted reaction between hemoglobin and a tetrazolium compound.
However, in a method for quantitatively determining a specific component in a biological specimen, which comprises reacting a biological specimen, in the presence of an electron acceptor, with an enzyme which has an ability, by the dehydrogenation reaction, to oxidize a specific component in a biological specimen or a substance derived from the specific component, and measuring the formed reductant of the electron acceptor, it is necessary to take into consideration not only the influence of change of the absorption wavelength of hemoglobin or influence of reducing properties thereof, but also inhibition of electron transport system. Accordingly, the influence of hemoglobin can not sufficiently be avoided even by the above-mentioned methods.